The objective of this project is to identify small molecule probes that selectively activate a chymotrypsin-like serine protease zymogen. Although zymogens are widely processed by proteolytic removal of a propeptide, this mechanism of activation is difficult to control in biological systems. Consequently, the biological functions of many proteases remain obscure. Here we propose to screen the NIH small molecule library collection to identify small molecule probes that activate a chymotrypsin-like serine protease (CSP) zymogen, without the requirement for proteolytic processing of the proenzyme. A high-throughput screening assay for CSP activating compounds has been developed and optimized using resonance energy transfer. Compounds that exhibit high potency selectivity over related family members, and structurally similar proteases will be downselected in secondary screening assays. Non-proteolytic zymogen activation will be verified using orthogonal assays. The best hits downselected from secondary screens will be subjected to two to three rounds of medicinal chemistry to further improve their potency and selectivity. The chemical probes arising from this work will be applied to study how serine proteases can be activated, and to investigate the functions of CSP in tumor metastasis and synaptic plasticity.